Fertilization is central to medically important problems of reproductive biology such as infertility and contraception, as well as the normal development of the human embryo. A better understanding of the entry and movements of the spermatozoon, the mechanism responsible for the activation of the metabolism of the unfertilized egg, and the block to polyspermy will lead to new research approaches for treating infertility, chemical contraception and problems during embryonal development. The aims of this three year proposal are to investigate the molecular and ionic mechanisms of egg activation, the incorporation of the spermatozoon, the movement and fusion of the male and female pronuclei and the late block to polyspermy. Specifically the objectives are to discover: 1) the role of the outer surface of the egg, external calcium and the spermatozoon in triggering the sequence of activation using pH changes and morphological criteria; 2) the sites of sequestrations and resequestration, and conditions for release of the internal calcium ions and protons by the use of fluorescent indicators and cell fractionation; 3) the consequences of increased cytoplasmic pH after fertilization by blocking or altering this pH change after insemination; 4) the activity of microfilaments in the egg cortex during the rotation and detachment of the spermatozoon as observed from the cortical surface by electron microscopy; 5) the formation, isolation, directionality and calcium storage ability of the sperm aster, and the splitting and separation of the sperm centriole studied in the live egg with a new method for observing these events; and 6) the mechanism of the late block to polyspermy as a function of sperm binding or membrane changes. These investigations will fill the gaps in parts of the knowledge about the early events at fertilization and will provide the framework on which to investigate other problems of fertilization and cell biology.